CN101481669B - Preparation and use of lilac grey streptomycete and active product thereof - Google Patents

Abstract

The present invention belongs to the technical field of biological pesticides, discloses a Streptomyces lavendulae xjy which has been collected in the general microorganism center of CCCCM in 8.16.2007 with the collection number: CCMCC No.2130. The present invention also discloses a separation and authentication method for the strain, a preparation method for antibiotic active substances of the xjy strain, authentication for analysis structures of the active substances, prevention and cure experiments for metabolic active substances of the xjy strain against leaf muld of tomato, powdery mildew of cucumber and phytophthora blight of pepper, and growth-promoting experiments for the growth of the strain. The antibiotic active substances secreted by the xjy strain is mainly nucleosides antibiotic having the characteristics of persistent effect and no medicinal hazard as well as having the effects of obviously preventing and curing vegetable diseases and promoting the growth to a certain extent.

Description

A kind of preparation method and application of Streptomyces lilacinus griseus and its active product

technical field

The invention belongs to the technical field of biopesticides, and in particular relates to a preparation method and application of a novel Streptomyces lilacinus griseus and its active product.

Background technique

Due to the long-term use of chemical pesticides, plant diseases have adaptive variation to fungicides. Under the long-term selection pressure of some fungicides, obvious resistance can occur, resulting in the failure of chemical control. The application strategies of different fungicides should be considered comprehensively in production, so as to give full play to the effects of various fungicides and avoid the emergence of drug resistance as much as possible. Since Waksman and Henrici established the genus Streptomyces in 1943, Streptomyces has become the genus with the largest number of actinomycetes and the largest variety of antibiotics, with more than 1,000 species and varieties.

As an important group of biopesticides, agricultural antibiotics are more efficient, faster, and safer than other biopesticides, and they all play an important role in the control of crop diseases and insect pests. However, the current market share of biopesticides in my country is only 2-3%, and its potential is far from being fully developed and applied.

Contents of the invention

The purpose of the present invention is to search for a new source of biopesticides, and utilize soil microbial resources to develop a broad-spectrum, high-efficiency, industrially-producible Streptomyces lilacinus griseus and a preparation method for active products that can be used for the prevention and treatment of plant fungal diseases .

The concrete technical scheme that realizes above-mentioned object of the present invention is as follows:

A Streptomyces lavendulae xjy, (hereinafter referred to as xjy strain), was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on August 16, 2007, and its abbreviation is CGMCC, and the preservation center registered in The book number is CGMCC No.2130, and the depository address is: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101.

The xjy bacterial strain of the present invention is that 6 strains have inhibitory activity to tomato leaf mold (Fulvia fulva) from 51 strains of soil actinomycetes screened out of cotton field soil in Xinjiang, China, wherein the inhibitory effect of bacterial strain xjy is the best. The antimicrobial spectrum of the xjy strain was measured, and it was found that its sterile fermentation filtrate had different degrees of inhibitory effect on 23 common plant pathogenic fungi and 6 kinds of bacteria, and the xjy strain had good antibacterial stability and could produce β-1,3- Glucanase and protease. The xjy strain has the following characteristics: ①The aerial hyphae on Gao’s No. 1 medium are lavender-gray, and become gray over time, and the hyphae in the base are light yellow, and no soluble pigment is produced; Long stalk, cylindrical spores, smooth surface, often forming long spore chains on air filaments; ③ Physiological and biochemical reactions: can liquefy gelatin, coagulate milk, hydrolyze starch, and produce hydrogen sulfide; and can grow on cellulose, But does not decompose filter paper; can utilize glucose, sucrose, maltose, fructose, lactose, starch, inositol, rhamnose and sorbose. ④The 16S rDNA sequence analysis results were compared with the maximum homology in GeneBank, and it was found that the 16S rDNA sequencing results had a 99.6% homology with the 16S rDNA sequence of Streptomyces lilacinus griseus.

Through the analysis of the morphological characteristics, culture characteristics, physiological and biochemical characteristics, cell wall components and 16S rDNA homology sequence analysis of the xjy strain, it was confirmed that it was Streptomyces lilacinus.

Another object of the present invention is to provide the sterile supernatant of the xjy bacterial strain, which is prepared by the following method: at first the seed culture medium (every liter containing 5g peptone, 3g yeast extract, 2g glucose, 1g beef extract, pH7.0 , liquid volume 40mL/250mL) at 28 ℃, 80r/min shaking culture 2d to make seed liquid; then add 10% seed liquid in the fermentation medium (containing soybean powder 2%, glucose 2%, NaCl 0.8%, CaCO ₃ 0.2%, (NH ₄ ) ₂ SO ₄ 0.32%, pH 7.0, liquid volume 40mL/250mL) at 28°C, 180r/mim shake flask culture for 6 days, the fermentation broth was centrifuged twice at 13000rpm, each time for 20min, namely Obtain a sterile supernatant.

Still another object of the present invention is to provide the antibacterial active substance of xjy bacterial strain, have following chemical formula C ₁₄ H ₁₉ O ₇ N ₃ and the chemical structural formula represented by (I):

The inventors found that the active substance produced by the bacterial strain xjy has good thermal stability, acid-base stability and light ultraviolet stability through the stability measurement of the sterile supernatant after fermentation of the bacterial strain xjy, and is alkaline water-soluble substance. Use oxalic acid to adjust the pH value of the sterile supernatant of the xjy strain to 2 to precipitate overnight. After suction filtration, use an equal volume of acetone to incubate in a water bath at 50°C for 1 hour. After filtering and concentrating to remove the acetone, adjust the pH to 7, and filter to obtain the pre- Antibacterial active substances are treated; the sterile fermentation filtrate is separated by pretreatment, activated carbon adsorption method, ion exchange method, resin adsorption method, and silica gel column chromatography to obtain the crude product of the active substance; it is purified by gel column and reverse phase column chromatography to obtain The pure product of the active substance; the determination of the physical and chemical properties of the pure product of the active substance found that the pure product is soluble in water, insoluble in general organic solvents, positive in the α-naphthol reaction test, ninhydrin reaction test and fuchsin reaction test, melting point It is 200-204°C, and the specific rotation is [α]D=+22.3°. Through the determination and analysis of ultraviolet absorption spectrum, infrared absorption spectrum, mass spectrum and nuclear magnetic resonance spectrum, the molecular weight of the pure product is finally deduced to be 341, the molecular formula is C ₁₄ H ₁₉ O ₇ N ₃ and the chemical structural formula.

The analysis of the antibacterial active substance components of the xjy bacterial strain of the present invention is obtained by technologies such as pretreatment of the active substance, separation of the crude product, and extraction of the pure product. From the analysis of the infrared spectrum, the pure product should contain -COOH, -NH ₂ , >C=C<, CH ₂ , -CH ₃ , -C=O, COC, COOR and glycopeptide structural groups. From the analysis of ¹³ C nuclear magnetic resonance carbon spectrum, ¹ H nuclear magnetic resonance hydrogen spectrum and two-dimensional map analysis, it is concluded that the molecule should contain 14 carbon atoms, including one carbonyl carbon atom, five hexose sugar atoms, and five carbon atoms on the carbon atom. The resonant peaks connecting the amino group and the oxygen connected to the carbon atom were identified as nucleoside antibiotics.

Still another object of the present invention is to provide the application of Streptomyces lilacaceae xjy strain in the prevention and treatment of tomato leaf mold, cucumber powdery mildew and pepper blight.

Another object of the present invention is to provide the sterile supernatant of Streptomyces lavender gray strain xjy for use in the prevention and treatment of tomato leaf mold, cucumber powdery mildew and pepper blight.

It is used in the control of tomato leaf mold, cucumber powdery mildew and pepper blight.

Still another object of the present invention is to provide the application of the antibacterial active substance (I) of Streptomyces lilacaceae xjy in promoting the growth of tomato and pepper.

The advantages and effects of the present invention: it has a stable source of natural raw materials, the strains are derived from soil, and the screening method is simple; the raw materials used in the fermentation process mainly include soybean powder and glucose, and the cost is low; the preparation of antibacterial active substances is simple, easy to operate, and the product Stable quality, good stability to light, heat, acid and alkali. The material has no phytotoxicity to crops, is green and environmentally friendly, and has a good effect of preventing and controlling vegetable diseases.

Description of drawings

Fig. 1 is a diagram of the inhibitory effect of antibacterial active substances of the xjy bacterial strain of the present invention on several plant pathogenic bacteria.

Figure 2 is a morphological diagram of the xjy strain of the present invention.

Fig. 3 is a graph showing thermal stability results of antibacterial active substances of the xjy strain of the present invention.

Fig. 4 is the chromatogram of the eluate of the antibacterial active substance of the xjy strain of the present invention on a polar macroporous resin.

Figure 5 is the elution profile of the antibacterial active substances of the xjy strain of the present invention on the gel column.

Fig. 6 is the infrared spectrogram of the pure product of the antibacterial active substance of the xjy bacterial strain of the present invention.

Fig. 7 ¹³ C-NMR chart of the pure product of the antibacterial active substance of the xjy strain of the present invention.

Fig. 8 ¹ H- ¹ H COZY diagram of the pure product of the antibacterial active substance of the xjy strain of the present invention.

Fig. 9 is the HSQC chart of the pure product of the antibacterial active substance of the xjy bacterial strain of the present invention.

Fig. 10 is a structural diagram of the pure product of the antibacterial active substance of the xjy strain of the present invention.

Fig. 11 is a diagram of greenhouse control of cucumber powdery mildew by antibacterial active substances of xjy strain of the present invention.

Fig. 12 is the greenhouse control diagram of the antibacterial active substance of xjy bacterial strain of the present invention to pepper blight.

Fig. 13 is a diagram of the field control effect of the antibacterial active substance of the xjy strain of the present invention on tomato leaf mold.

Fig. 14 is a diagram of the growth-promoting action of pepper plant growth of the antibacterial active substance of the xjy strain of the present invention.

Detailed ways

The following examples are used to further describe the present invention, but the content of the present invention is not limited thereto.

Embodiment one: Screening and identification of bacterial strain xjy

Through the antibacterial test in the dish, 6 strains were screened out from 51 strains of soil actinomycetes to have inhibitory activity against tomato leaf mold (Fulviafulva), among which strain xjy had the best inhibitory effect. Determination of the antibacterial spectrum of strain xjy found that its sterile fermentation filtrate had different degrees of inhibitory effect on 23 common plant pathogenic fungi and 6 kinds of bacteria (Figure 1, A: tomato leaf mold B: wheat sheath blight C: kiwi fruit dry Bacteria D: pear rot pathogen.), and the strain xjy has good antibacterial stability in subculture, and can produce β-1,3-glucanase and protease; through the morphological characteristics of the strain xjy (Fig. 2A: spores; B: hyphae), culture characteristics, physiological and biochemical characteristics, cell wall component analysis and 16S rDNA homology sequence analysis, it was determined that the strain xjy was Streptomyces lilacinus.

Embodiment two: the preparation method of bacterial strain xjy antibacterial active substance

First, vibrate on the seed medium (5g peptone per liter, 3g yeast extract, 2g glucose, 1g beef extract, pH7.0, liquid volume 40mL/250mL) at 28°C, 80r/min for 2 days to make seed liquid; then Add 10% seed solution to the fermentation medium (per liter contains 2% soybean powder, 2% glucose, 0.8% NaCl, 0.2% CaCO ₃ , 0.32% (NH ₄ ) ₂ SO ₄ , pH 7.0, liquid volume 40mL /250mL) at 28°C and 180r/mim shake flask culture for 6 days, and the fermentation liquid was centrifuged twice at 13000rpm for 20min each time to obtain the sterile supernatant.

Embodiment three: the property and structural analysis of the active substance produced by bacterial strain xjy

Through the stability measurement of the sterile supernatant after fermentation of strain xjy, it was found that the active substance produced by strain xjy had good thermal stability (Fig. 3, ①A: 50°C for 30min; ①B: 50°C for 60min; ①C: 50°C 90min; ①D: 120min at 50°C; ①E: 150min at 50°C; ①F: sterile supernatant; ②A: 30min at 100°C; ②B: 60min at 100°C; C: 90min at 100°C; 150min; F: sterile supernatant), acid-base stability, and light-UV stability, which are alkaline water-soluble substances; the fermented sterile supernatant of xjy strain was adjusted to pH 2 with oxalic acid to precipitate overnight, and suction filtered Afterwards, use an equal volume of acetone to incubate in a water bath at 50°C for 1 h, filter and concentrate to remove the acetone, then adjust the pH to 7, and obtain the pretreated antibacterial active substance by filtering; For elution, collect the test tubes with biological activity and combine them, and concentrate them to a small volume by rotary evaporation; then load the sample on 732 cation exchange resin, elute with 0.2mol/L ammonia water, collect the test tubes with biological activity and combine them, and concentrate them by rotary evaporation Use the 732 resin concentrate as a sample to load on the polar resin, first elute it with absolute ethanol and discard it, then elute it with deionized water, collect the water effluent and concentrate it to obtain a concentrated solution used as a weak The loading sample of the polar resin is carried out the chromatographic detection of various eluents by thin-layer chromatography at the same time, and the developing agent is chloroform: ethanol: water: n-butanol (8: 12: 2: 16), and the result It shows that a large amount of impurities can be removed by eluting with absolute ethanol first (Fig. 4, A: sample solution; B: absolute ethanol eluent; C: water eluent); the water-washed concentrate of the polar column is loaded In a weakly polar resin, and then eluted with 20% ethanol, the eluate collected and concentrated was loaded on a silica gel column. : 16: 8) for elution, and then eluted with water, and the water washing liquid was dried to obtain the crude product; the concentrated solution of the silica gel column was loaded on the Superdex peptide 10/300GL gel column of Amersham Company, and eluted with ultrapure water , at a wavelength of 280nm, the absorbance is detected, the collection tubes of each peak are combined and freeze-dried, and it is found that the color of the 8 peaks of the elution changes from brown to yellow, and the color gradually becomes lighter, and the 8th peak of the elution is an active peak ( Figure 5, AH: 8 elution peaks). The concentrated solution of the used gel column is loaded on the Resource RPC type reversed-phase column of Amersham Company, and carried out gradient elution with 0%A～100%A, A is an aqueous solution containing 1% formic acid, and B is methanol: acetonitrile (4 : 1), the absorption value was detected at a wavelength of 280nm, and the results were combined and dried in 4 sections to detect the activity, and the color of the dried sample gradually became darker. After testing, it was found that only peak No. 1 was active, and the active collection solution was combined and concentrated to load again. With acetonitrile: water: methanol (4:96:24) for elution, there are only two elution peaks after elution, of which 1 elution peak is active, combined, concentrated and dried to obtain a nearly white pure product; the physical and chemical properties of the pure active substance It is found that the pure product is soluble in water and insoluble in common organic solvents, and it is positive in the α-naphthol reaction test, ninhydrin reaction test and magenta reaction test, with a melting point of 200-204°C and a specific rotation of [α ] D = +22.3°. Through the determination _and analysis of ultraviolet absorption spectrum (Fig _. 6), infrared absorption spectrum, mass spectrum and _nuclear magnetic resonance spectrum ( _Fig . Structural formula (Figure 10);

Embodiment four: the potted control test of bacterial strain xjy active substance to tomato leaf mold, cucumber powdery mildew and capsicum blight

Greenhouse live inoculation method was adopted, three treatment methods were adopted for inoculation of pathogenic bacteria after spraying in advance, simultaneous spraying for inoculation of pathogenic bacteria and spraying after inoculation of pathogenic bacteria. The nutrient solution (i.e. the fermentation medium) is a spraying control, and a blank control (do not do any treatment) is established at the same time, and the test is repeated 3 times. The control test finds that the active substance produced by the bacterial strain xjy is effective against tomato leaf mold (Table 1), cucumber Powdery mildew (Fig. 11, A: spraying water control after inoculation; B: the control effect of spraying 10 times dilution of sterile supernatant before inoculation) and pepper blight (Fig. 12, A: spraying water control after inoculation; B : the control effect of spraying 10 times dilution of sterile supernatant before inoculation) all have better control effect, but the control effect to different diseases is different to some extent. The measurement of the control effect of sterile fermentation filtrate with different dilutions showed that the control effect decreased with the increase of dilution; the measurement of different application times showed that the control effect of spraying in advance was better than that of spraying after inoculation and spraying at the same time. Effect: The control effect of the crude product and carbendazim on tomato leaf mold, and the control effect of the crude product and metalaxyl on cucumber powdery mildew showed that the control effect of the crude product at the same concentration was better than that of the two pesticides, and had a better effect;

Table 1 Greenhouse Control Effect of Sterile Filtrate on Tomato Leaf Mold

Embodiment five: the field prevention and control test of bacterial strain xjy active product to tomato leaf mold

Adopt the prevention and control test of field natural disease investigation tomato leaf mold, find that the aseptic culture filtrate of bacterial strain xjy has good prevention and control effect to tomato leaf mold (Fig. 13, A: contrast (the first sub-district); B : the preventive effect of spraying 5 times dilution of sterile supernatant after the onset (the first plot); C: contrast (the third plot); D: the control effect of spraying 5 times dilution of sterile supernatant after the onset (third cell).). Under the same concentration, the prevention and control effect decreases with the decrease of the number of sprays; under the same number of sprays, the prevention and control effect also decreases with the increase of the dilution (table 2);

Table 2 Field control effect of culture filtrate on tomato leaf mold

Embodiment six: the promoting action of bacterial strain xjy active substance to tomato and capsicum

Tomato seedlings and pepper seedlings were treated by root irrigation, and root irrigation with clear water and soybean powder nutrient solution (ie, fermentation medium) was used as a control, and the experiment was repeated 3 times. It was found that the sterile culture filtrate not only promoted the growth of tomato plants and the growth of roots (Table 3), but also promoted the growth of pepper plants, and the effect increased with the increase of the concentration of the culture filtrate (Figure 14).

Table 3 The influence of culture filtrate on the growth of tomato plants

Claims (2)

1. A strain of Streptomyces lavender, named as Streptomyces lavendulae xjy, has been preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures. The preservation date is August 16, 2007, and the registration number is It is CCMCC No.2130. 2. the streptomyces lavender gray xjy bacterial strain described in claim 1 is used in the prevention and treatment of tomato leaf mold, cucumber powdery mildew and capsicum blight.

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